The Tetracyclines

7.3. The Tetracyclines

The epoch-making discovery of chlortetracycline (aureomycin) in 1947 by Duggar paved the way for a number of structural analogues used as broad-spectrum antibiotic that belong to the tetracy- cline family. The tetracyclines which are found to be effective therapeutically are listed in the following table.

7.3.1. Salient Features of the Tetracyclines

HC 3 CH 3 R 4 R 3 R 2 R 1 N

6 5 OH

8 7 6a C 5a 4a D 4 3

1 0a

11 a

9 10 11 12 12a

OH

C O N -R 3

OH O

OH O

ANTIBIOTICS

249 Name of

R 1 R 2 R 3 R 4 R 5 Compound

Official Status

Brand Name(s)

Tetracycline (R) BPC ; Tetracyn

H OH CH 3 H H (1973) ; USP ;

(Pfizer) SK-Tetracycline (R) (SK & F)

Oxytetracycline USP ;

Terramycin (R)

OH OH CH 3 H H

(Pfizer)

Chlortetracycline BP, USP ; Eur. P. ; Aureomycin (R)

H HCl

H OH CH 3 Cl

Int. P. ; Ind. P. ;

(Lederle)

Demeclocycline BP. USP ;

H HCl

Ledermycin (R)

H OH

H Cl

Eur. P. ;

(Lederle, UK)

Methacycline BP (1973) ;

OH = CH 2 H H HCl

Rondomycin (R)

USP ;

(Wallace)

Doxycycline USP ;

Vibramycin (R)

OH H CH 3 H H

(Pfizer)

Rolitetracycline USP ;

Syntetrin (R)

H OH CH 3 H—CH 2 —N

(Bristol)

7.3.2. Nomenclatures

Based on the above conventional numbering of various carbon atoms and subsequent labelling of the four aromatic rings present in the tetracycline nucleus, oxytetracycline is chemically designated as :

‘‘4-Dimethylamino-1, 4, 4a, 5, 5a, 6, 11, 12a-octahydro-3, 6, 10, 12, 12a-penta-hydroxy-6-me- thyl-1, 11-dioxo-2-naphthacenecarboxamide’’.

Some other members of the tetracycline family may conveniently be named as follows : Methacycline : 6-Methylene-5-oxytetracycline ; Doxycycline : α -6-Deoxy-5-oxytetracycline ; Rolitetracycline : N-(Pyrrolidinomethyl)-tetracycline.

7.3.3. General Characteristics of the Tetracyclines

Following are the general characteristic features of all the members of the tetracycline family : (a) The tetracycline are obtained by fermentation procedures from streptomyces species or by

the chemical transformations of the natural products. (b) The important members of this family are essentially derivatives of an octahydronaphthacene,

i.e., a hydrocarbon made up of a system of four-fused rings. (c) The antibiotic spectra and the chemical properties of these compounds are quite similar but

not identical.

PHARMACEUTICAL BIOTECHNOLOGY

(d) The tetracyclines are amphoteric compounds, i.e., forming salts with either acids or bases. In neutural solutions these substances exist mainly as zwitter ions.

(e) The acid salts of the tetracyclines that are formed through protonation of the dimethylamino groupf of C-4, usually exist as crystalline compounds which are found to be very much soluble in water. However, these amphoteric antibiotics will crystallize out of aqueous solu- tions of their salts unless they are duly stabilized by an excess of acid.

(f) The corresponding hydrochloride salts are used commonly for oral administration and are usually encapsulated owing to their bitter taste.

(g) The water soluble salts are obtained either from bases such as sodium/potassium hydroxides or formed with divalent/polyvalent metals, e.g., Ca ++ . The former ones are not stable in aqueous solutions, while the latter ones, e.g., calcium salt give tasteless products that may be employed to prepare suspensions for liquid oral dosage forms.

(h) The unusual structural features present in the tetracyclines afford three acidity constants (pKa values) in aqueous solutions of the acid salds. The thermodynamic pKa values has been extensively studied by Lesson et al. and discussed in the chapter on ‘Physical-chemical fac- tors and biological activities’.

(i) An interesting property of the tetracyclines is their ability to undergo epimerizaton at C-4 in

solutions having intermediate pH range. These isomers are called epitetracyclines. The four epi-tetracyclines have been isolated and characterized. They exhibit much less,

activity than the corresponding ‘natural’ isomers ; thus accounting for an apparent de- crease in the therapeutic value of aged solution.

CONH 2 CONH 2

e p i (le s s a c tive ) N a tu r a l (m o re a c tiv e ) (j) It has been observed that the strong acids and bases attack the tetracyclines having a hydroxy

moiety at C-6, thereby causing a considerable loss in activity through modification of the C- ring as shown below :

CONH 2 OH CONH 2 OH OH O

OH

O A n h y d ro tetra c y c lin e

OH O

Is o te trac y c lin e

(IN A C T IV E )

Strong acids produce a dehydration through a reduction involving the OH group at C-6 and the H atom at C-5a. The double bond thus generated between positions C-5a and C-6 in-

duces a shift in the position of the double bond between the carbon atoms C-11 and C-11a thereby forming the relatively more energetically favoured resonant system of the naphtha- lene group found in the inactive anhydrotetracyclines.

The strong bases on the other hand promote a reaction between the hydroxyl group at C-6 and the carbonyl moiety at C-11, thereby causing the bond between C-11 and C-11a atoms to cleave and eventually form the lactone ring found in the inactive isotetracyclines.

(k) The tetracyclines form stable chelate complexes with many metals, e.g., Ca ++ , Mg ++ , Fe ++ , etc.

A few typical examples for the tetracyclines shall be dealt with in the sections that follows :

2-Naphthacenecarboxamine [4S-(4 α , 4a α , 5a α ,6 β , 12a α )]-4-(dimethylamino)-1, 4, 4a, 5, 5a, 6,

11, 12a-octahydro-3, 6, 10, 12, 12a-pentahydroxy-6-methyl-1-11-dioxo- ; USP ; Achromycin (R) ; Cyclopar (R) ; Panmycin (R) ; Tetracyn (R) ;

PHARMACEUTICAL BIOTECHNOLOGY

Tetracycline* is the drug of choice in the treatment of chloera, relapsing fever, granuloma inguinale and infections produced by rickettsia, Borrelia, Mycobacterium fortuitum and marinum, and Chlamydia psittaci and trachomatis (except pneumonia and inclusion conjunctivitis).

It may be employed as an ‘alternative drug’ in the following two situations, namely : (a) With silver nitrate in the prevention of neonatal ocular prophylaxis of chlamydial and

gonococcal conjunctivitis, and (b) For treatment of actinomycosis, anthrax, chancroid, mellioidosis, plague, rat-bite fevers, syphi-

lis and yaws. It has also been reported to be beneficial in the treatment of toxoplasmosis.

Tetracycline Production

Tetracycline is produced on large-scale using the submerged fermentation process by two pre- dominant strains employed across the globe, namely : (a) Streptomyces aureofaciens [ATCC : 13908- 13911 and NCL B-9114]. In actual practice, the stock cultures are adequately maintained for reasonably long durations in the shape of spores. Importantly, the resulting spores are maintained strictly either

under liquid N 2 (– 70 to – 80°C) or lyphilized.** The ‘bioreactor’ being employed is normally made up of stainless-steel along with SS connecting pipes, two-or three way SS gate-valves, SS-pumps (Alpha Laval - Make) provided with adequate agitation and compressed sterile air circulation. Besides, it must have all the necessary gadgets and recording devices meant for round-the-clock monitoring during the entire fermentative operation. Various physical and physiological parameters of the culture media in the bioreactor need to be controlled automatically, such as : pH regulation, supplementation with sterile nutrients during the fermentation run etc. In addition, a number of vital and critical tests are being carried out for the optimum growth of the antibiotic (tetracycline), for instance : strength of nutrients, morphology and growth of culture, antibiotic production, and sterility conditions.

Note : Sterilization of the ‘liquid nutrient media’ is normally carried out at 120°C for a period of 40 minutes.

Culture Medium : It has been observed that the overall tetracycline production is solely gov- erned by the ensuing C : N ratio of the nutrients (i.e., sources) in the culture medium. However, in actual practice the various components that essentially provide carbon sources are, namely : starch, sucrose and glycerol ; and nitrogen sources are, namely : admixture of soyabean meal plus mineral salts, ammonium salts, amino acids, casein (milk-protein), meat-extract (animal-protein) etc. Besides, the usable medium also comprises of cotton-seed meal, peanut meal, cornsteep liquor etc. It is absolutely important as well as necessary to stringently maintain very low concentration of Cl – ion in the medium so as to accomplish high production levels. Deionized cornsteep liquor and similar raw materials free from Cl – ions may also be used gainfully. The optimum temperature should be 28°C and pH must vary between 5.5 to 6.5 (ideally 5.6 to 6.0).

Inoculum : The basic inherent characteristic features of the ensuing inoculum do play a major role for the biosynthetic production of tetracycline, such as : quality of vegetative inoculum or spores i.e., its threshold age, genetic homogeneity, metabolic reative profile etc. Optimum tetracycline yield is duly accomplished from a medium that predominantly comprises of inoculum for 24 hours, and present within a range of 2-10% having an optimum pH value between 5.6 and 6.2.

* Kar, A., Medicinal Chemistry, New Age International Publishers, New Delhi, 3rd. edn., 664-667, 2005. ** Rapid freezing of a substance at an extremely low temperature and then dehydrating the substance in a high

ANTIBIOTICS

Aeration : It is quite necessary and equally important to make available both vigorous and inten- sive aeration in the submerged cultures of Streptomyces aureofaciens right from the very initial stage of cultivation phenomenon. Any observed irregular aeration or interruption in aeration during the first-

few hours invariably give rise to an appreciable extent of production of tetracycline.

Tetracycline Production : The teracycline production is carreid out in submerged aerated agi- tated bioreactor containing adequate virulent culture medium loaded with genetically homogenous and metabolically reactive inoculum. It essentially comprise of three distinct and vital stages, namely :

Stage-1 : Growth Phase : It is mostly characterized by instant fast utilization of incorporated nutrients. There is a distinct enhancement of the cell mass. The phsophate (PO –3 4 ) ion concentration has an enormous influence upon the prevailing culture medium. Interestingly, during the on-going produc- tion phase of the tetracycline fermentative procedure the secondary mycelium (i.e., the thin-hyphae) is found to modulate the specific phosphate ions present in the culture medium. However, the ‘production

type of the mycelium’ is hardly generated particularly in the overwhelmingly presence of the PO –3 4 ions. Stage-2 : Production Phase : In this particularly phase the maximum quantum of the antibiotic

is actually generated. Thus, the overall rate of growth of the concerned microorganism gets decreased substantially and almost ceases in due course.

State-3 : Thus is, in fact, the last phase wherein the production of the tetracycline almost attains the lowest ebb. The mycelium undergoes due fragmentation and the process of cleavage commences apparently.

Isolation and Purification : Tetracycline is invariably obtained from the clear filtrate ob- tained from the acidic medium (i.e., the fermented broth) by the help of sterilized SS-Plate Type Filter Press or Pdobielniak Counter Current Extractor (as mentioned under ‘penicillin’). The clear filtrate is subjected to a process of adsorpton upon an active substance e.g., activated carbon, and elution subse- quently. The eluted liquid is treated with a requisite amount of salts of alkaline earth metals to obtain the precipitate of tetracycline as its corresponding salts. The resulting salt is poorly water soluble, but fairly soluble in several organic solvents. Once the organic phase is separated, the tetracycline is ad- equately pushed into the aqueous phase (by the addition of diluted HCl). Finally, the purified form of

tetracycline is salted out or crystallised carefully, and dried under vacuo.